Genetic analysis of connectivity in neural networks (S. Roux, F. Robin, C. Saint Cloment, J. Miana Mena
We have constructed several fusion proteins between tetanos toxin fragments and a reporter gene, LacZ or GFP. These molecules retains retrograde intracellular and trans-synaptic transports properties We have shown that the intramuscular transport depends critically on neural endogenous activity, implying by analogy that in central synapses transcellular transfer requires also activity in the pre-synaptic cell .Simple transgenics animals have been constructed and analysed. Cell specific promotors like calbindin and L7, as well as an ubiquitous promotor, CMV, have been used. Our results in vivo have established the approach's feasibility using a combination of two reporter genes. We can identify during embryogenesis cells in which transcription occurs and connected cells receiving reporter proteins by an activity dependent transport .We are analyzing connectivity in various genotypic context by constructing animals with a genetic conditional triggering of the reporter genes synthesis. The dynamic progression of the reporter protein inside the neural network as well as the details of the intracellular transport will be monitored by multiphoton confocal microscopy, eventually when possible in living animals. Paramagnetic proteins detectable by fMRI and sensitive to neural activity are being studied .
Calcium imaging in neural networks (V. Baubet, H. Le Mouellic )
Calcium is an universal second messenger with critical roles at various levels of neural information processing. By analogy with the fluorescing jellyfish in response to calcium influx, we have constructed new calcium sensitive bioluminescent proteins by fusing aequorin and GFP . The main advantage of this new calcium imaging is that we can target the gene to intracellular organelles, to specific receptors and channels, as well as specific neural subpopulations in transgenics animals. In addition, optical microscopy allows to detect long range correlation of calcium fluctuations in different cells of a tissue. We have already targeted such a reporter gene to the Hoxc-8 locus in a transgenic animal and to the pre-synaptic apparatus by fusion with synaptotagmin. We are planning to target the reporter protein to the endoplasmic reticulum and to active dendrites so as to follow, in transgenics animals, the intracellular modifications of neuronal informations during its processing and successive integration from a dendritic compartment up to eventual modifications of gene expression in the nuclei. A new and powerful imaging system will be used to follow in real time small calcique fluctuations.
Genetic mechanims of brain development (R. Hashemi, S. Picaud)
Null mutant mice with Otx1 or Otx2 homeogenes replaced by LacZ were created. By 9.5 dpc homozygous Otx2-/-mutant embryos are characterized by the lack of brain structures anterior to rhombomere 3. Otx2 expression at the onset of gastrulation is required for neural induction. Gene expression profiles in WT and mutant embryos were compared by SAGE at E6.5. Among a broader list , the study of Otx2 downstream genes allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the gastrulating embryos. A conditional inactivation of FGF 15, an Otx2 target, is undertaken .Otx1-/- mice show a later phenotype,epileptic behavior and multiple brain abnormalities. Otx1 mutant mice are also defective in the refinement of long distance, exuberant axonal projections, suggesting that Otx1 is required for the development of normal axonal connectivity and the generation of coordinated motor behavior .
Hox Homeogenes and innervation (H. Le Mouellic)
Mice deficient for the homeotic gene HoxC8 display homeotic transformations of mesodermal derivatives. Innervation of forelimb distal muscles is also abnormal. HoxC8 expressing motoneurons in spinal segments C7 to T1 undergo increased apoptosis at E13.5 during the neuromuscular junction's formation. Somatotopic maps are also altered along the rostrocaudal and the mediolateral axis.To identify Hox homeogenes upstream genes, a new genetic method is being developed relying on an exhaustive mutagenesis of ES cell genome by a new retroviral vector.
Reprogramming somatic cells into stem cells (S. Picaud)
In the last few years, live births have been achieved using somatic nuclear transfer in mice, sheep, cows and goats and the technique would probably also be successful in human. The greatest benefit of the new technology will likely be in therapeutic cloning ; the use of somatic cell nuclear transfer to generate replacement tissues or organs. This would avoid the risks of tissue rejection by supplying a person with new tissues of exactly their own genetic type. The overall effect of transferring a somatic nuclei into an egg is to reboot its genetic programme for embryogenesis. Yet unidentified factors localized into the egg cytoplasm can reprogram genes in a coordinated fashion. Knowing that a developmental program can be rebooted, we explore if one can genetically reprogram somatic cells into stem cells and identify the factors involved.